Cystatins are a family of inhibitors of papain-like (family C1) and legumain-related (family C13) cysteine peptidases such as mammalian cathepsins B, H and L. Four main cystatin families have been described: type-I cystatins (stefins) are primarily cytoplasmic, type-II cystatins are secreted inhibitors and are single-domain proteins, type-III cystatins (kininogens), and type-IV cystatins (fetuins). Cystatin D is a Type II cystatin that also include Cystatin C and S-type (S, SN, and SA). Cystatin D was initially cloned from a genomic library using a cystatin C cDNA probe. Cystatin C is ubiquitously expressed; Cystatin D and S-Type are in saliva and share a high degree of amino acid similarity. Cystatin C possesses higher activity than Cystatin D and S-type. In addition, Cystatin D is unable to inhibit cathepsin B and shows a preferential inhibition of cathepsin S over cathepsins H and L.Product Details
- Human Cystatin D amino acids (Gln26 - Val 142) (Accession # P28325) was expressed in CHO cells.The carboxy terminus contains TG-8 His tag.
- Molecular Mass
- The 123 amino acid recombinant protein has a predicted molecular mass of approximately 14.3 kD. The DTT-reduced and non-reduced protein migrate at approximately 17 kD and 15 kD respectively by SDS-PAGE. The predicted amino terminal is Gln.
- > 95% by SDS-PAGE gel as determined by Coomassie stained SDS-PAGE.
- 0.22 µm filtered protein solution is in 20 mM MES, 0.15 M NaCl, pH 6.0
- Endotoxin Level
- Less than 1.0 EU per µg cytokine as determined by the LAL method.
- 10-25 µg sizes are bottled at 200 µg/mL. 100 µg and larger size are bottled at the concentration indicated on the vial.
- Storage & Handling
- Unopened vial can be stored between 2°C and 8°C for up to 2 weeks, at -20°C for up to six months, or at -70°C or colder until the expiration date. For maximum results, quick spin vial prior to opening. The protein can be aliquoted and stored at -20°C or colder. Stock solutions can also be prepared at 50 - 100 µg/mL in appropriate sterile buffer, carrier protein such as 0.2 - 1% BSA or HSA can be added when preparing the stock solution. Aliquots can be stored between 2°C and 8°C for up to one week and stored at -20°C or colder for up to 3 months. Avoid repeated freeze/thaw cycles.
- The activity of human Cystatin D is measured by its property to inhibit human Papain (0.5 µg/mL) activity when 100 µM of Z-FR-AMC is used as a papain peptide substrate. The IC50 value is <8 nM.
- Application Notes
BioLegend carrier-free recombinant proteins provided in liquid format are shipped on blue-ice. Our comparison testing data indicates that when handled and stored as recommended, the liquid format has equal or better stability and shelf-life compared to commercially available lyophilized proteins after reconstitution. Our liquid proteins are validated in-house to maintain activity after shipping on blue ice and are backed by our 100% satisfaction guarantee. If you have any concerns, contact us at email@example.com.
Human Cystatin D Assay Procedures
Human Cystatin D activity is measured by its ability to inhibit cleavage of a fluorogenic peptide substrate Z-Phe-Arg-AMC by papain protease. When papain cleaves the substrate, the increase of the product is monitored by increase in intensity of fluorescent emission at 460 nm with excitation at 380 nm.
Materials and Buffers
- Activation Buffers: pH 7.0, 50 mM Tris, 5 mM DTT.
- Assay Buffers: pH 7.0, 50 mM Tris.
- Human Cystatin D: Biolegend
- Papain substrate: Z-FR-AMC (20 mM in DMSO)
- Activate papain in ice cold activation buffer at 100 ug/mL for 15 minutes.
- Prepare 1:2 serial dilution of human Cystatin D from the top concentration of 1200 nM in Assay Buffer.
- Prepare 2 ug/mL papain solution in activation buffer with activated papain from step 1.
- Add 50 uL of papain (prepared in step 3) to 50 ul of human cystatin D serial dilutions (prepared in step 2).
- Incubate the mixtures from number 4 for 15 minutes at room temperature.
- Prepare the substrate to 200 µM in Assay Buffer.
- Transfer 50 µL of each mixture solution to a black bottom plate.
- Add 50 µL of 200 µM of the substrate solution into each well to start the reaction.
- Read the reaction progress at 380/460 nm (Excitation/Emission) in kinentic mode for 5 minutes.
- The final human Papain concentration is 0.5 µg/mL (50 ng) and the substrate concentration is 100 µM. The top concentration of human Cytatin D is 300 nM in the assay.
Highly expressed in salivary gland, submandibular and parotid glands.
- Cystatin D demonstrates a preferential inhibition of cathepsin S > cathepsin H > cathepsin L and no inhibition of cathepsin B or pig legumain. Possible proteinase showing protective function in the oral cavity. Induced by vitamin D in colon cancer cells.
- Cathepsin S, Cathepsin H, Cathepsin L
- Cystatin D inhibits human Papain activity.
- Biology Area
- Cancer Biomarkers, Cell Biology
- Molecular Family
- Enzymes and Regulators
- Antigen References
- Freije JP, et al. 1991. J. Biol. Chem. 266:20538-43.
- Alvarez-Fernández M, et al. 2005. J. Biol. Chem. 280:18221-8.
- Alvarez-Díaz S, et al. 2009. J. Clin. Invest. 119:2343-58.
- Dickinson DP, et al. 2002. DNA Cell. Biol. 21:47-65.
- de Sousa-Pereira P, et al. 2014. PLoS One. 9(10):e109050.
- Hill LJ, et al. 2017. Sci. Rep. 7:5002.
- Gene ID
- 1473 View all products for this Gene ID
- View information about Cystatin D on UniProt.org
- Does specific activity of a recombinant protein vary between lots?
Specific activity will vary for each lot and for the type of experiment that is done to validate it, but all passed lots will have activity within the established ED50 range for the product and we guarantee that our products will have lot-to-lot consistency. Please conduct an experiment-specific validation to find the optimal ED50 for your system.
- Have your recombinants been tested for stability?
Our testing shows that the recombinant proteins are able to withstand room temperature for a week without losing activity. In addition the recombinant proteins were also found to withstand four cycles of freeze and thaw without losing activity.
- How do you convert activity as an ED50 in ng/ml to a specific activity in Units/mg?
- Use formula Specific activity (Units/mg) = 10e6/ ED50 (ng/mL)
- How does the activity of your recombinant proteins compare to competitors?
We quality control each and every lot of recombinant protein. Not only do we check its bioactivity, but we also compare it against other commercially available recombinant proteins. We make sure each recombinant protein’s activity is at least as good as or better than the competition’s. In order to provide you with the best possible product, we ensure that our testing process is rigorous and thorough. If you’re curious and eager to make the switch to BioLegend recombinants, contact your sales representative today!
- What is the specific activity or ED50 of my recombinant protein?
The specific activity range of the protein is indicated on the product datasheets. Because the exact activity values on a per unit basis can largely fluctuate depending on a number of factors, including the nature of the assay, cell density, age of cells/passage number, culture media used, and end user technique, the specific activity is best defined as a range and we guarantee the specific activity of all our lots will be within the range indicated on the datasheet. Please note this only applies to recombinants labeled for use in bioassays. ELISA standard recombinant proteins are not recommended for bioassay usage as they are not tested for these applications.